Effects of Monensin on Ethanol-Induced Alterations in Function of Hepatocellular Asialoglycoprotein Receptor Subpopulations

Abstract

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 microM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.

Ethanol and Membrane Protein Trafficking: Diverse Mechanisms of Ethanol Action

“…We have extensively studied the process of RME by the hepatocyte-specific asialoglycoprotein receptor (ASGP-R) and have identified multiple steps of this pathway that are affected by ethanol treatment (Casey et al, 1987(Casey et al, , 1990Tworek et al, 1996Tworek et al, , 1997Tworek et al, , 1998b. We have extensively studied the process of RME by the hepatocyte-specific asialoglycoprotein receptor (ASGP-R) and have identified multiple steps of this pathway that are affected by ethanol treatment (Casey et al, 1987(Casey et al, , 1990Tworek et al, 1996Tworek et al, , 1997Tworek et al, , 1998b.…”

Receptor‐mediated endocytosis by the asialoglycoprotein receptor: effect of ethanol administration on endosomal distribution of receptor and ligand

“…Our previous investigations, utilizing the same ethanol‐feeding model used in the current study, have shown that ethanol impairs RME by ASGP‐R. The ethanol‐induced impairments in RME, which include ligand binding, internalization and degradation, receptor recycling, endosomal acidification, and receptor hyperphosphorylation, were investigated in both isolated hepatocytes and isolated perfused rat liver (1–3, 5, 12–17). In the current study we administered the labeled ligand and allowed endocytosis to proceed in vivo , and then processed the whole liver to isolate endosomes.…”

“…For the receptor, it seems that the receptor-containing endosomes are not being properly recycled and/or are being transported farther into the cell than that observed under normal conditions. Based on previous work in our laboratory, we knew that exposure of rats to ethanol resulted in impaired receptor-ligand dissociation (2,12,13); further, we observed impaired endosomal acidification in ethanol-fed rats compared with the controls using a fluid-phase marker, FITC-Dextran (4). In order to determine if the ethanol-induced shift in ligand and ASGP-R distribution between the two different endosome populations resulted from impaired receptorligand dissociation, we assessed the B/F ligand ratios in the endosome fractions.…”

Ethanol and Membrane Protein Trafficking: Diverse Mechanisms of Ethanol Action

“…Ethanol administration impairs hepatic protein metabolism at several steps along the protein transport pathway, including the cellular process of RME. We have extensively studied the process of RME by the hepatocyte-specific asialoglycoprotein receptor (ASGP-R) and have identified multiple steps of this pathway that are affected by ethanol treatment (Casey et al, 1987(Casey et al, , 1990Tworek et al, 1996Tworek et al, , 1997Tworek et al, , 1998b. The ASGP-R is an abundant endocytic receptor that is predominantly expressed on the sinusoidal surface of hepatocytes and has been well studied both in isolated hepatocytes and cultured liver cells of both human and rat origin.…”